Comparison of the levels of infectious virus in respirable aerosols exhaled by ferrets infected with influenza viruses exhibiting diverse transmissibility phenotypes

Abstract

Influenza viruses pose a major public health burden to communities around the world by causing respiratory infections that can be highly contagious and spread rapidly through the population. Despite extensive research on influenza viruses, the modes of transmission occurring most often among humans are not entirely clear. Contributing to this knowledge gap is the lack of an understanding of the levels of infectious virus present in respirable aerosols exhaled from infected hosts. Here, we used the ferret model to evaluate aerosol shedding patterns and measure the amount of infectious virus present in exhaled respirable aerosols. By comparing these parameters among a panel of human and avian influenza viruses exhibiting diverse respiratory droplet transmission efficiencies, we are able to report that ferrets infected by highly transmissible influenza viruses exhale a greater number of aerosol particles and more infectious virus within respirable aerosols than ferrets infected by influenza viruses that do not readily transmit. Our findings improve our understanding of the ferret transmission model and provide support for the potential for influenza virus aerosol transmission.

Fig 1

Comparison of morbidity and influenza virus titers in ferret nasal washes. Ferrets were inoculated intranasally (IN) or by aerosol (AR) inhalation with 10^2.7 to 10⁷ PFU of virus and were monitored daily for 6 days (n = 4 [SI06, MX09, and BD11], 5 [PN99], and 6 [TH04]). (A) Mean weight loss ± standard deviation (SD) is shown as the percentage of baseline for each virus group. (B) Mean minute volume of respiration ± SD was measured on alternating days in sedated animals by whole-body plethysmography and is shown as the percentage of baseline for each virus group. (C) Mean virus titers ± SD in nasal wash samples collected on alternating days are shown. No TH04-infected ferrets survived past day 4. Circles containing “IN” or “AR” represent the amount of inoculum administered to ferrets by the respective method of inoculation.

Fig 2

Analysis of aerosols exhaled by individual influenza virus-infected or uninfected ferrets. Particle size distributions of aerosols exhaled by ferrets inoculated with 10^2.7 to 10⁷ PFU of virus IN (green lines) or by AR inhalation (orange lines) or by naive animals (black lines) during normal breathing (A) or sneezing stimulation (B) are shown (n = 4 [SI06, MX09, and BD11], 5 [PN99], 6 [TH04], and 11 [naive]). Collection procedures were performed at 2, 4, and 6 days after inoculation, but these time points are not distinguished. Total particle counts in the aerodynamic diameter range of 0.5 to 20 μm are shown, but due to space constraints, selected sizes are noted on the x axis.

Fig 3

Analysis of aerosol volumes exhaled by influenza virus-infected and uninfected ferrets. The total aerosol volume exhaled (not the volume of air) from naive ferrets (triangles) or from ferrets inoculated with 10^2.7 to 10⁷ PFU of virus IN (green) or by AR inhalation (orange) during normal breathing or sneezing (n = 4 [SI06, MX09, and BD11], 5 [PN99], 6 [TH04], and 11 [naive]) is shown. Total volumes were determined for aerosols 0.5 to 20 μm (A), 0.5 to 5 μm (B), and 0.5 to 1 μm (C) in size exhaled from individual animals. Collection procedures were performed at 2, 4, and 6 days after inoculation, but these time points are not distinguished. Mean values ± SDs are shown. The statistical significance of comparisons between human and avian influenza virus groups is shown; ns, not significant.

Fig 4

Rates of recovery of influenza virus. The rates of recovery of infectious influenza virus using the viable cascade impactor were assessed by spiking the collection medium on impactor plates with virus (10¹ to 10⁵ PFU) and then passing sterile air through the impactor for 30 or 5 min. Virus titers in collection medium were determined by plaque assay without dilution and compared to the amount used to spike the plate. The mean percentage (+SD) of recovery for each virus is shown.

Fig 5

Infectious virus present in aerosols exhaled from influenza virus-infected ferrets. Aerosols were collected from ferrets inoculated with 10^2.7 to 10⁷ PFU of PN99 (A), SI06 (B), MX09 (C), TH04 (D), or BD11 (E) virus IN (green) or by AR inhalation (orange) during normal breathing or sneezing using a viable cascade impactor (n = 4 [SI06, MX09, and BD11], 5 [PN99], and 6 [TH04]). Virus titers collected on impactor plates on alternating days after inoculation were determined by plaque assay without dilution, and total PFU is shown for each ferret on the left y axis. Viral RNA levels in the same samples from combined IN and AR experiments were estimated by real-time RT-PCR and are shown as lines on the right y axis.